Resistance to currently available antibiotics has created a need for new antibiotic agents. Infections, caused by organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecium and Enterococcus faecalis, have become increasingly resistant to currently approved antibiotics. For example, significant clinical problems include methicillin-resistant strains of S. aureus, which are resistant to all current antibiotics except vancomycin (a drug of last resort because of severe side effects), and a vancomycin-resistant strain of E. faecium enterococci, which is now found world-wide. Even community-acquired organisms such as Streptococcus pneumoniae are increasingly resistant to antimicrobial agents, with a significant number of isolates being resistant to penicillin and extended-spectrum cephalosporins.
The emergence and spread of resistant bacterial organisms are primarily caused by acquisition of drug resistance genes, resulting in a broad spectrum of antibiotic resistance (e.g., extended-spectrum cephalosporin-resistant mutant xcex2-lactamases found in several bacterial organisms). Genetic exchange of multiple-resistance genes, by transformation, transduction and conjugation, combined with selective pressures in settings such as hospitals where there is heavy use of antibiotic therapies, enhance the survival and proliferation of antimicrobial agent-resistant bacterial strains occurring by, e.g., spontaneous mutation. Although the extent to which bacteria develop resistance to antimicrobial drugs and the speed with which they do so vary with different types of drugs, resistance has inevitably developed to all antimicrobial agents (see, Gold and Moellering, Jr., 1996, New Eng. J Med., 335(19):1445-1453).
To prevent or delay the buildup of a resistant pathogen population, different chemicals that are effective against a particular disease-causing bacterium must be available. Thus, there is a need to identify compounds which can penetrate and specifically kill the pathogenic bacterial cell, or arrest its growth without also adversely affecting its human, animal, or plant host.
One avenue for accomplishing this task involves the use of compounds targeting bacterial RNA polymerase. Accordingly, what is needed in the art are new compounds which are effective inhibitors of bacterial RNA polymerase and useful as antimicrobial agents. The present invention provides such compounds along with methods for their use.
In a first aspect, the present invention provides compounds having the formula: 
wherein R1 is selected from H, ORxe2x80x2 and NRxe2x80x2Rxe2x80x3, wherein Rxe2x80x2 and Rxe2x80x3 are independently selected from H and substituted or unsubstituted lower alkyl. R2 and R3 are independently selected from substituted or unsubstituted aryl, substituted or unsubstitued heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted heteroaryl-heteroalkyl, and substituted or unsubstituted aryl-heteroalkyl.
Unless otherwise indicated, the compounds provided in the above formulas are meant to include pharmaceutically acceptable salts and prodrugs thereof.
In another aspect, the present invention provides pharmaceutical compositions comprising a compound of formula I or II in admixture with a pharmaceutically acceptable carrier or excipient.
In still another aspect, the present invention provides methods for treating or preventing bacterial growth in a subject by administering to the subject a therapeutically effective amount of a compound of formula I or II.
In yet another aspect, the present invention provides methods for modulating bacterial growth on a surface comprising contacting the surface with a compound of formula I or II.
Other objects and advantages of the present invention will be apparent from the detailed description that follows.
Abbreviations and Definitions
The abbreviations used herein are conventional, unless otherwise defined.
The term xe2x80x9calkyl,xe2x80x9d by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multi-valent radicals, having the number of carbon atoms designated (i.e. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)ethyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term xe2x80x9calkyl,xe2x80x9d unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below as xe2x80x9cheteroalkyl,xe2x80x9d xe2x80x9ccycloalkylxe2x80x9d and xe2x80x9calkylene.xe2x80x9d The term xe2x80x9calkylenexe2x80x9d by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by xe2x80x94CH2CH2CH2CH2xe2x80x94. Typically, an alkyl group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A xe2x80x9clower alkylxe2x80x9d or xe2x80x9clower alkylenexe2x80x9d is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
The terms xe2x80x9calkoxy,xe2x80x9d xe2x80x9calkylaminoxe2x80x9d and xe2x80x9calkylthioxe2x80x9d refer to those groups having an alkyl group attached to the remainder of the molecule through an oxygen, nitrogen or sulfur atom, respectively. Similarly, the term xe2x80x9cdialkylaminoxe2x80x9d is used in a conventional sense to refer to xe2x80x94NRxe2x80x2Rxe2x80x3 wherein the R groups can be the same or different alkyl groups.
The term xe2x80x9cacylxe2x80x9d or xe2x80x9calkanoylxe2x80x9d by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and an acyl radical on at least one terminus of the alkane radical.
The term xe2x80x9calkoxycarbonylxe2x80x9d denotes xe2x80x94C(O)OR wherein R is alkyl as defined herein.
The term xe2x80x9calkylcarbamoylxe2x80x9d denotes xe2x80x94C(O)NRxe2x80x2Rxe2x80x3 wherein Rxe2x80x2 and Rxe2x80x3 are independently selected alkyl groups as defined herein.
The term xe2x80x9csulfonylxe2x80x9d denotes xe2x80x94SO2xe2x80x94.
The term xe2x80x9csulfamoylxe2x80x9d denotes xe2x80x94SO2NH2.
The term xe2x80x9cheteroalkyl,xe2x80x9d by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyl group. The heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples include xe2x80x94CH2xe2x80x94CH2xe2x80x94Oxe2x80x94CH3, xe2x80x94CH2xe2x80x94CH2xe2x80x94NHxe2x80x94CH3, xe2x80x94CH2xe2x80x94CH2xe2x80x94N(CH3)xe2x80x94CH3, xe2x80x94CH2xe2x80x94Sxe2x80x94CH2xe2x80x94CH3, xe2x80x94CH2xe2x80x94CH2xe2x80x94S(O)xe2x80x94CH3, xe2x80x94CH2xe2x80x94CH2xe2x80x94S(O)2xe2x80x94CH3, xe2x80x94CHxe2x95x90CHxe2x80x94Oxe2x80x94CH3, xe2x80x94Si(CH3)3, xe2x80x94CH2xe2x80x94CHxe2x95x90Nxe2x80x94OCH3, and xe2x80x94CHxe2x95x90CHxe2x80x94N(CH3)xe2x80x94CH3. Up to two heteroatoms may be consecutive, such as, for example, xe2x80x94CH2xe2x80x94NHxe2x80x94OCH3 and xe2x80x94CH2xe2x80x94Oxe2x80x94Si(CH3)3. Also included in the term xe2x80x9cheteroalkylxe2x80x9d are those radicals described in more detail below as xe2x80x9cheteroalkylenexe2x80x9d and xe2x80x9cheterocycloalkyl.xe2x80x9d The term xe2x80x9cheteroalkylenexe2x80x9d by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by xe2x80x94CH2xe2x80x94CH2xe2x80x94Sxe2x80x94CH2CH2xe2x80x94 and xe2x80x94CH2xe2x80x94Sxe2x80x94H2xe2x80x94CH2xe2x80x94NHxe2x80x94CH2xe2x80x94. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini. Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied.
The terms xe2x80x9ccycloalkylxe2x80x9d and xe2x80x9cheterocycloalkylxe2x80x9d, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of xe2x80x9calkylxe2x80x9d and xe2x80x9cheteroalkylxe2x80x9d, respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like.
The terms xe2x80x9chaloxe2x80x9d or xe2x80x9chalogen,xe2x80x9d by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as xe2x80x9cfluoroalkyl,xe2x80x9d are meant to include monofluoroalkyl and polyfluoroalkyl.
The term xe2x80x9caryl,xe2x80x9d employed alone or in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwise stated, an aromatic substituent which can be a single ring or multiple rings (up to three rings), which are fused together or linked covalently. xe2x80x9cHeteroarylxe2x80x9d are those aryl groups having at least one heteroatom ring member. Typically, the rings each contain from zero to four heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. The xe2x80x9cheteroarylxe2x80x9d groups can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl ring systems are selected from the group of acceptable substituents described below. The term xe2x80x9carylalkylxe2x80x9d is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) or a heteroalkyl group (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).
Each of the above terms (e.g., xe2x80x9calkyl,xe2x80x9d xe2x80x9cheteroalkylxe2x80x9d and xe2x80x9carylxe2x80x9d) are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.
Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from, for example: xe2x80x94ORxe2x80x2, xe2x95x90O, xe2x95x90NRxe2x80x2, xe2x95x90Nxe2x80x94ORxe2x80x2, xe2x80x94NRxe2x80x2Rxe2x80x3, xe2x80x94SRxe2x80x2, -halogen, xe2x80x94SiRxe2x80x2Rxe2x80x3Rxe2x80x2xe2x80x3, xe2x80x94OC(O)Rxe2x80x2, xe2x80x94C(O)Rxe2x80x2, xe2x80x94CO2Rxe2x80x2, CONRxe2x80x2Rxe2x80x3, xe2x80x94OC(O)NRxe2x80x2Rxe2x80x3, xe2x80x94NRxe2x80x3C(O)Rxe2x80x2, xe2x80x94NRxe2x80x2xe2x80x94C(O)NRxe2x80x3Rxe2x80x2xe2x80x3, xe2x80x94NRxe2x80x3C(O)2Rxe2x80x2, xe2x80x94NHxe2x80x94C(NH2)xe2x95x90NH, xe2x80x94NRxe2x80x2C(NH2)xe2x95x90NH, xe2x80x94NHxe2x80x94C(NH2)xe2x95x90NRxe2x80x2, xe2x80x94S(O)Rxe2x80x2, S(O)2Rxe2x80x2, xe2x80x94S(O)2NRxe2x80x2Rxe2x80x3, xe2x80x94CN and xe2x80x94NO2 in a number ranging from zero to (2N+1), where N is the total number of carbon atoms in such radical. Rxe2x80x2, Rxe2x80x3 and Rxe2x80x2xe2x80x3 each independently refer to hydrogen, unsubstituted (C1-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When Rxe2x80x2 and Rxe2x80x3 are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, xe2x80x94NRxe2x80x2Rxe2x80x3 is meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term xe2x80x9calkylxe2x80x9d is meant to include groups such as haloalkyl (e.g., xe2x80x94CF3 and xe2x80x94CH2CF3) and acyl (e.g., xe2x80x94C(O)CH3, xe2x80x94C(O)CF3, xe2x80x94C(O)CH2OCH3, and the like).
Similarly, substituents for the aryl groups are varied and are selected from: -halogen, xe2x80x94ORxe2x80x2, xe2x80x94OC(O)Rxe2x80x2, xe2x80x94NRxe2x80x2Rxe2x80x3, xe2x80x94SRxe2x80x2, xe2x80x94Rxe2x80x2, xe2x80x94CN, xe2x80x94NO2, xe2x80x94CO2Rxe2x80x2, xe2x80x94CONRxe2x80x2Rxe2x80x3, xe2x80x94C(O)Rxe2x80x2, xe2x80x94OC(O)NRxe2x80x2Rxe2x80x3, xe2x80x94NRxe2x80x3C(O)Rxe2x80x2, xe2x80x94NRxe2x80x3C(O)2Rxe2x80x2, xe2x80x94NRxe2x80x2xe2x80x94C(O)NRxe2x80x3Rxe2x80x2xe2x80x3, xe2x80x94NHxe2x80x94C(NH2)xe2x95x90NH, xe2x80x94NRxe2x80x2C(NH2)xe2x95x90NH, xe2x80x94NHxe2x80x94C(NH2)xe2x95x90NRxe2x80x2, xe2x80x94S(O)Rxe2x80x2, xe2x80x94S(O)2Rxe2x80x2, xe2x80x94S(O)2NRxe2x80x2Rxe2x80x3, xe2x80x94N3, xe2x80x94CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where Rxe2x80x2, Rxe2x80x3 and Rxe2x80x2xe2x80x3 are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl, (unsubstituted aryl)-(C1-C4)alkyl, (unsubstituted aryl)oxy-(C1-C4)alkyl and perfluoro(C1-C4)alkyl.
Two of the substituents on adjacent atoms of the aryl ring may optionally be replaced with a substituent of the formula xe2x80x94Txe2x80x94C(O)xe2x80x94(CH2)qxe2x80x94Uxe2x80x94, wherein T and U are independently xe2x80x94NHxe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94CH2xe2x80x94 or a single bond, and the subscript q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl ring may optionally be replaced with a substituent of the formula xe2x80x94Axe2x80x94(CH2)rxe2x80x94Bxe2x80x94, wherein A and B are independently xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)2xe2x80x94, xe2x80x94S(O)2NRxe2x80x2xe2x80x94 or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl ring may optionally be replaced with a substituent of the formula xe2x80x94(CH2)sxe2x80x94Xxe2x80x94(CH2)txe2x80x94, where s and t are independently integers of from 0 to 3, and X is xe2x80x94Oxe2x80x94, xe2x80x94NRxe2x80x2xe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94S(O)2xe2x80x94, or xe2x80x94S(O)2NRxe2x80x2xe2x80x94. The substituent Rxe2x80x2 in xe2x80x94NRxe2x80x2xe2x80x94 and xe2x80x94S(O)2NRxe2x80x2xe2x80x94 is selected from hydrogen or unsubstituted (C1-C6)alkyl.
As used herein, the term xe2x80x9cheteroatomxe2x80x9d is meant to include, for example, oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).
As used herein, the term xe2x80x9cmodulatexe2x80x9d means to reduce, prevent or otherwise, control, microbial growth. The microbes whose growth is modulated include bacteria, viruses, mycobacterium, yeasts and parasites. In preferred embodiments, the microbes are bacteria. The term modulate is meant to include effects that are both cidal and static.
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d is meant to include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge, S. M., et al., xe2x80x9cPharmaceutical Saltsxe2x80x9d, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.
The neutral forms of the compounds may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are substantially equivalent to the parent form of the compound for the purposes of the present invention.
In addition to salt forms, the present invention provides compounds which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent drug is not. The prodrug may also have improved solubility in pharmacological compositions over the parent drug. A wide variety of prodrug derivatives are known in the art, such as those that rely on hydrolytic cleavage or oxidative activation of the prodrug. An example, without limitation, of a prodrug would be a compound of the present invention which is administered as an ester (the xe2x80x9cprodrugxe2x80x9d), but then is metabolically hydrolyzed to the carboxylic acid, the active entity. Additional examples include peptidyl derivatives of a compound of the invention.
Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.
Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are all intended to be encompassed within the scope of the present invention.
Certain compounds of the invention may exist in one or more tautomeric forms. The present invention encompasses the various tautomeric forms of the compounds of the invention, including both single tautomers and mixtures of tautomers.
The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (3H), iodine-125 (125I) or carbon-14 (14C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
Compounds
In one aspect, the present invention provides compounds of the formula 
wherein R1 is selected from H, ORxe2x80x2 and NRxe2x80x2Rxe2x80x3, wherein Rxe2x80x2 and Rxe2x80x3 are independently selected from H and substituted or unsubstituted lower alkyl. R2 and R3 are selected from substituted or unsubstituted aryl, substituted or unsubstitued heteroaryl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, heteroarylalkyl, substituted or unsubstituted heteroaryl-heteroalkyl, and substituted or unsubstituted aryl-heteroalkyl. Preferably, when R2 is a halo-susbstituted phenyl group, R3 is other than a phenyl group substituted with a moiety bound to said phenyl group via a sulfur atom.
In a preferred embodiment, R2 and R3 are independently selected from substituted or unsubstituted aryl and substituted or unsubstitued heteroaryl groups. In this embodiment, the substituted aryl and substituted heteroaryl groups are preferably substituted with a member selected from, hydroxyl, halogen, nitro, cyano, substituted or unsubstituted (C1-C6)alkyl, (C1-C6)heteroalkyl, (C1-C6)alkoxy, (C1-C6)alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, xe2x80x94C(O)mR4, xe2x80x94C(O)NR4R5, xe2x80x94S(O)nR4, xe2x80x94SO2NR4R5, xe2x80x94NR4R5, xe2x80x94NR6C(O)mxe2x80x94R4, xe2x80x94NR6C(O)NR4R5, xe2x80x94NR6S(O)nR4, xe2x80x94OC(O)mR4 and xe2x80x94OC(O)NR4R5, wherein m is an integer independently selected from 1 and 2 and n is independently selected from the numbers from 0 to 2. R4, R5 and R6 are independently selected from hydrogen, substituted or unsubstituted (C1-C8)alkyl, substituted or unsubstituted (C1-C8)heteroalkyl or one or more of R4, R5, and R6 is substituted or unsubstituted (C3-C6)alkyl or substituted or unsubstituted (C3-C6)heteroalkyl combined with the nitrogen atom to which it is attached to form a four-, five-, six- or seven-membered ring optionally having additional substituents selected from substituted or unsubstituted (C1-C8)alkyl, substituted or unsubstituted (C1-C8)heteroalkyl and substituted or unsubstituted phenyl.
In another preferred embodiment, the substituted aryl groups are substituted phenyl groups.
In another preferred embodiment, one or both of R2 and R3 are selected from: 
in which, R7, R8 and R9 are independently selected from hydroxyl, halogen, nitro, cyano, substituted or unsubstituted (C1-C6)alkyl, (C1-C6)haloalkyl, substituted or unsubstituted (C1-C6)heteroalkyl, (C1-C6)alkoxy, (C1-C6)haloalkoxy, (C1-C6)alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, alkanoyl, alkoxycarbonyl, alkylcarbamoyl, sulfonyl, sulfamoyl and NR4R5, wherein R4 and R5 are defined as above.
In each of the above R7, R8 and R9 groups, the alkyl portions of, for example, (C1-C6)alkyl, (C1-C6)alkylthio and (C1-C6)alkoxy may be further substituted with, for example, one or more halogen, hydroxy, nitro, cyano group, etc.
In a further preferred embodiment, R2 and R3 are independently selected from: 
wherein X is a halogen, preferably Cl or F, and R14 is selected from hydrogen, substituted or unsubstituted (C1-C8)alkyl, C(O)mR4, C(O)NR4R5, S(O)nR4, SO2NR4R5, NR4R5, NR6C(O)mR4, NR6C(O)NR4R5, NR6S(O)nR4, OC(O)mR4 and OC(O)NR4R5. R4, R5, R6 and m and n are defined as above.
In yet another preferred embodiment, the present invention provides compounds of formula (III): 
in which, R10, R11, R12 and R13 are independently selected from H, halogen and haloalkyl groups, with the proviso that at least two of R10, R11, R12 and R13 are groups are other than H. In this embodiment, the preferred halogen groups are chloro and fluoro groups and particularly preferred distributions of the chloro and fluoro groups results in a first compound in which R10, R11 and R13 are chloro or fluoro groups and a second compound in which R10, R12 and R13 are chloro or fluoro groups.
In another preferred embodiment, the compounds of the invention have the formula (IV): 
in which, R10, R11, R12 and R13 are independently selected from H, halogen and haloalkyl groups. In a still further preferred embodiment, R11 and R13 are H. In yet another preferred embodiment, R10 is xe2x80x94CF3 and R12 is Cl or F.
In yet another preferred embodiment, the substituted heteroaryl groups contain one heteroatom selected from N, O and S.
In a further preferred embodiment, R2 and R3 are independently selected from: 
wherein R14 is defined as above.
Synthesis of Pyrazoles and Related Derivatives
Compounds of the present invention can be prepared using readily available materials or known intermediates. Schemes 1 and 2 provide exemplary synthetic routes for the production of selected compounds of the invention. One of skill in the art will understand that additional methods are also useful. 
In Scheme 1, an aryl derivative (i) with an active hydrogen (e.g., arylacetonitrile) is condensed with an aryl aldehyde under phase transfer conditions to form the corresponding benzylidene (ii). The benzylidene derivative is subsequently reacted with an agent, such as lithium trimethylsilyldiazomethane to form the desired cyclized adduct, which is subjected to a basic reaction milieu to produce a substituted pyrazole (iii).
Scheme 2 provides an exemplary route to selected pyrazolone-based compounds of the invention. 
In Scheme 2, an aryl derivative (iv) with an active hydrogen and a leaving group (e.g. ethyl ester) is deprotonated with a base, such as n-butyllithium, and coupled to an activated aryl carbonyl derivative, such as an acyl halide. The resulting substituted ketone (v) is reacted with a nitrogen donor, such as hydrazine in an acidic milieu, thereby forming a substituted pyrazolone (vi).
Regarding the molecular structures set forth in Schemes 1 and 2, one of skill in the art will readily appreciate that precursor and intermediates having substituents other than phenyl derivatives, e.g., heteroaryl derivatives such as thiophene derivatives, can be used to practice the synthetic route. Moreover, it will be appreciated that the groups R and Rxe2x80x2 indicate, in a very general sense, substituents on the aryl groups. R and Rxe2x80x2 can be the same or different. Both R and Rxe2x80x2 can represent a single substituent or multiple substituents. When R and/or Rxe2x80x2 represent multiple substituents, each R and Rxe2x80x2 can be the same or different.
Methods of Using the Compounds as Antimicrobial Agents
The compounds of invention are preferably inhibitors of RNA polymerase activity. In a preferred embodiment, the compounds of the invention have an IC50 against a RNA polymerase of from about 0.1 xcexcM to about 250 xcexcM, more preferably from about 1 xcexcM to about 100 xcexcM. The IC50 values of the compounds of the invention can be determined using art-recognized assays, such as that set forth in Example 39.
Still further preferred compounds inhibit the growth and reproduction of microorganisms (e.g., bacteria, viruses, mycobacterium, yeasts, and parasites). Thus, certain preferred compounds will interact with a microorganism with a minimum inhibitory concentration of from 1 nM to about 250 xcexcM, more preferably from about 50 nM to about 100 xcexcM, and even more preferably from about 1 xcexcM to about 10 xcexcM. The minimum inhibitory concentration (MIC) of the compounds of the invention can be determined using art-recognized assays, such as those set forth in Example 39. The spectrum of inhibition of the compounds of the invention, i.e., the range of microorganisms whose growth and reproduction are inhibited by the compounds of the invention, may be narrow, broad or extended, as determined in a standard test system.
In another preferred embodiment, the compounds of the invention are used to modulate the growth of microorganisms on a surface. As used herein, a surface refers generally to a wide range of objects, including, for example, household, industrial and hospital surfaces (e.g., fixtures, floors, linens). Also included are surfaces, such as tissues (e.g., skin, mucosal), and organs (e.g., ocular). When the surface is a tissue or organ, the compounds of the invention, in this embodiment, will generally be administered topically and are useful when administered in vivo, in vitro and ex vivo.
In another preferred embodiment, the compounds of the invention are used to reduce, retard or prevent a microbial infection in a subject. In this embodiment, the subject is treated with an amount effective to reduce, retard or prevent the infection.
Evaluation of Compounds as Antimicrobial Agents
The compounds of the present invention can be evaluated for antimicrobial activity in a variety of assay formats known to those of skill in art. The specific assays used to select the most appropriate compound for use will typically depend on the targeted microorganism or infection.
One common assay involves evaluation of the compounds as RNA polymerase inhibitors. In this assay, buffer (250 mM KCl, 5% glycerol, 10 mM MgCl2, 0.1 mg/ml BSA) is combined with 6 mM xcex2-M.E., PT5 DNA template, and 1.3 xcexcg/rxn Sigma70 saturated E. coli RNA Polymerase (Epicenter). The compound is then added in a manner not to exceed 5% DMSO. Nucleotide triphosphates are then added at the following concentration: 250 xcexcM ATP, CTP and UTP with 100 xcexcM cold CTP and 50 xcexcM xcex1-32P CTP. The mixture is incubated for 10 min at about 37xc2x0 C. A [2xc3x97] loading buffer is added and the mixture is then run on a 6% urea denaturing PAGE until bromophenol blue reaches the edge of plate. The gel is soaked (about 20 minutes in 10% MeOH and 10% acetic acid, to remove urea), then dried (about 55 minutes at about 85xc2x0 C. (BioRad Gel Drier)) and exposed to a Phospho Imaging Plate for 1 hour. The plate is then read on a Fujix Bas1000 Imaging System and quantified using MacBas v2.0 software. An IC50 (in xcexcM) can be calculated as the concentration of a drug which reduces the enzyme activity to 50% of the control.
For Minimum Inhibitory Concentration (MIC) determinations for selected bacteria, log phase growing bacteria are re-suspended at 1xc3x97105 bacteria per mL in LB medium. The compound is added and two-fold dilutions are made. The final volume in the 96-well plate is about 100 xcexcL. The plate is incubated at 37xc2x0 C. in the dark with shaking. After 16 hours of incubation, growth is monitored by reading A600 or by visual inspection. MIC is defined as the minimum concentration of drug resulting in inhibition of visible growth of bacterial under the conditions described (above) in National Committee for Clinical Laboratory Standards 1993. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7-A3; National Committee for Clinical Laboratory Standards: Villanova, Pa.
Formulations and Administration of Antimicrobial Agents
The compounds of the present invention can be prepared and administered in a wide variety of oral, topical and parenteral dosage forms. Thus, the compounds of the present invention can be administered by injection, that is, intravenously, intramuscularly, intracutaneously, subcutaneously, intraduodenally, or intraperitoneally. The present invention also contemplates the administration of the compounds of the present invention in a depot formulation. Also, the compounds described herein can be administered by inhalation, for example, intranasally. Additionally, the compounds of the present invention can be administered transdermally. Accordingly, the present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier or excipient and either a compound of formula I, II or a pharmaceutically acceptable salt or prodrug thereof.
For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
The powders and tablets preferably contain from 5% or 10% to 70% of the active compound. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term xe2x80x9cpreparationxe2x80x9d is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active component is dispersed homogeneously therein, as by stirring. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.
The pharmaceutical preparation is preferably in unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
The quantity of active component in a unit dose preparation may be varied or adjusted from 0.1 mg to 1000 mg, preferably 1.0 mg to 100 mg according to the particular application and the potency of the active component. The composition can, if desired, also contain other compatible therapeutic agents.
In therapeutic use for the treatment of bacterial infections, the compounds utilized in the pharmaceutical method of the invention are administered at the initial dosage of about 0.001 mg/kg to about 100 mg/kg daily. A daily dose range of about 0.1 mg/kg to about 10 mg/kg is preferred. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
In another preferred embodiment, the compounds and compositions of the invention are formulated to include or are used with other antimicrobial agents. In certain patient populations and with particular antimicrobial disorders, combination therapy results in increased efficacy over single-agent therapy. Combination therapy may also allow for the reduction in dosage of one or more of the agents used in combination therapy and, concomitantly, result in the reduction of adverse effects associated with each agent.
A wide range of antimicrobial agents can be used with the compounds, compositions and methods of the present invention. Such agents can be categorized based on their mechanism of action and/or their chemical structure or properties. For example, antimicrobial agents may act by interfering with cell wall synthesis, plasma membrane integrity, nucleic acid synthesis, ribosomal function, and folate synthesis. The compounds and compositions of the present invention may be used in conjunction with antimicrobial agents from each of these categories. In preferred embodiments, the compounds and compositions of the present invention are used in combination with antibiotics.
Agents that interfere with cell wall synthesis include the xcex2-lactams (e.g, penicillins (including, for example, penicillin V, penicillin G, amoxicillin, ampicillin, nafcillin, ticarcillin, carbenicillin, and cloxacillin) and cephalosporins (including, for example, cephalexin, cefoxitin, ceforanide, and cefaclor)), which inhibit peptidoglycan polymerization, and by vancomycin, which combines with cell wall substrates. Agents with interfere with plasma membrane integrity, causing leakage, include, the polymyxins (including, for example, polymyxin B and colistin). The plasma membrane sterols of fungi are targeted by polyenes such as amphotericin. Agents which affect nucleic acid synthesis include the quinolones (for example, ciprofloxacin and norfloxacin) which bind to a bacterial complex of DNA and DNA gyrase, thereby blocking DNA replication, and rifampin-related agents that block RNA synthesis by binding to DNA directed RNA polymerase. Agents that interfere with ribosomal function include the aminoglycosides (e.g., gentamicin, tobramycin and neomycin), tetracycline, chloramphenicol, the macrolides (e.g., erythromycin and clarithormycin) and clindamycin. The sulfonamides (sulfamethoxazole and sulfisoxazole) and trimethoprim represent agents involved in blocking the synthesis of the folate needed for DNA replication. Other agents suitable for combination therapy include biosurfactants (e.g., circulin, EM49, polypeptin, brecistin, cerexin, tridecephin, surfactin, subsporin, mycosubtilisin, bacillomycin), and miscellaneous antibiotics (e.g., capreomycin, bacitracin, gramicidin, gramicidin S, tyrocidine).
The following examples are offered by way of illustration and are not intended to either define or limit the scope of the invention.